Method for forming matrices of hardened material

ABSTRACT

A matrix of hardened material, typically biocompatible material, is formed by contacting a hardenable liquid with a volume blanking structure, the structure having a dispersion of interconnected spaces and including a hardening agent and allowing the hardenable liquid to harden by interaction with the hardening agent to form the matrix. The volume blanking structure may be removed to leave corresponding voids in the matrix of hardened material. The hardenable liquid may contain viable cells.

FIELD OF THE INVENTION

The present invention relates to the production of hardened material inthe form of a matrix, by hardening a hardenable liquid. The invention isparticularly applicable to the production of bulk matrices ofbiocompatible material, but is not limited to this.

DESCRIPTION OF THE PRIOR ART

There is a demand for replacement tissue for implantation into the humanbody. Typically, replacement blood vessels are required for use insurgical procedures (Vacanti and Langer (1999), Lancet 35A, Supplement1: pages 32 to 34). Also, there is a need for replacement tissue ofbulkier and/or more complex nature, such as body organs. Typicalexamples include organs such as the liver, kidneys and heart.

In the case, for example, of dual kidney failure, a patient faces theprospect of artificial dialysis until a suitable donor kidney is foundfor transplantation. Dialysis has the drawbacks of inconvenience and therisk of infection. Transplantation is often complicated by rejectionproblems. Even more severe complications are presented when the organwhich fails is the heart or liver.

There have been attempts to construct tissue engineering scaffolds usingbiocompatible materials. Typical scaffold materials are plasticsmaterials such as PGA (polyglycolic acid), PLA (polylactic acid) andPLGA (polylactic coglycolic acid). These materials are formed intodesired shapes by conventional techniques such as melting followed byextrusion or moulding. Since the plastics material must be melted beforeextrusion or moulding, there is limited opportunity to incorporatebiologically active molecules or cells in the shaped material.

Previous attempts to construct tissue engineering scaffolds haveinvolved the seeding and growth of cells on sheets or tubes ofbiocompatible material. Attempts to make bulk tissue engineeringscaffolds have involved lamination of sheets such as PGA to give athicker construction (see, for example, Mikos A G, Sarakinos G, Leite SM, Vacanti J P, and Langer R, Laminated three dimensional biodegradablefoams for use in tissue engineering, 1993 BIOMATERIALS 14; 323-330, andCima L G and Cima M J, 1996, Tissue regeneration matrices by solidfree-form fabrication techniques, U.S. Pat. No. 5,518,680). However,such techniques are complex and involve many steps of lamination. Thismakes these techniques unsuited to anything more than laboratory scaleexperimental use.

It is also known to encapsulate cells in materials such as alginate forimplantation into mammals, in order to achieve delivery of therapeuticmolecules secreted by the cells to a desired tissue (see for example T.A. Read et al, Nature Biotechnology 19, pages 29 to 34 and T. Joki etal, Nature Biotechnology 19, pages 35 to 39). In this case, cells aretypically encapsulated in beads of alginate.

It is also known to mix cells with collagen and allow the mixture toset. However, FDA-approved collagen is extremely costly (around $1 permicrogram) so this technique is unsuited to the formation of tissueengineering scaffolds of useful size.

WO-00/62829 describes manufacture of biocompatible porous polymerscaffolds by pouring a solution of polymer in two miscible solvents ontowater-soluble particles, then cooling to crystallise the polymer andremoving the solvents and the particles.

SUMMARY OF THE INVENTION

Accordingly, in a first aspect, the present invention provides a methodof forming a matrix of hardened material, including the steps of:

-   -   contacting a hardenable liquid with a volume blanking structure,        the structure having a dispersion of interconnected spaces        therein and including a hardening agent, whereby the hardenable        liquid occupies at least some of said spaces in said structure;        and    -   allowing the hardenable liquid to harden by interaction with the        hardening agent to form the matrix.

By “hardened material” is meant a material which is sufficiently hardsubstantially to retain its form or shape without the volume blankingarrangement, but it may sag to some extent. Matrices formed according tothe present invention will typically be flexible and preferably will beresilient. Indeed, matrices formed according to this invention may bedelicate. Thus the term “hardened material” is used to encompass, interalia, materials having a high liquid content such as hydrogels andreadily deformable and flexible materials. However, the formation ofmore rigid structures is also contemplated.

In this first aspect, the invention may be envisaged as providing a“negative” or mould for the final hardened matrix by the volume blankingstructure. The volume blanking structure may be envisaged as “blankingout” certain volumes to the hardenable liquid, i.e. excluding thehardenable liquid from those volumes. Thus, the hardenable liquid islocatable in the interconnected spaces but is excluded from theblanked-out volumes.

It is not necessary that the volume blanking structure is formed beforecontact is made with the hardenable liquid, although this is preferred.

Typically, the hardening interaction is a chemical interaction, such ascross-linking of molecules in the hardenable liquid. By a chemicalinteraction is meant typically a chemical reaction causing chemicalchange. A physical change only, e.g. crystallisation such as thecrystallisation procedure of WO-00/62829, does not constitute a chemicalinteraction. A suitable combination of hardenable liquid and hardeningagent can be chosen depending on the use to which the structure to beformed is to be put, and taking account of constraints imposed by othercomponents to be incorporated within the structure.

The method may further include the step of removing the volume blankingstructure to leave corresponding voids in the matrix of hardenedmaterial. Preferably, the volume blanking structure is removed bydissolving it. The matrix of hardenable material remaining is preferablyporous.

In the case where the matrix has a distribution of voids in it afterremoval of the volume blanking structure, the voids are preferablyinterconnected. This allows fluid (e.g. cell culture medium) to flowthrough the matrix, via the interconnected voids.

It is preferred that the matrix of hardened material is a bulk matrix.This is in contrast with the hardenable liquid being hardened into theform of a sheet or a tube. Preferably, the bulk matrix has a threedimensional shape whose smallest external overall dimension is not lessthan 0.5 mm, 1 mm, 2 mm, 5 mm or, more preferably 10 mm. Forming a bulkmatrix has the advantage that a tissue engineering scaffold can beformed essentially in one piece, rather than by layering of individualthin pieces of material. As is explained later, an advantage ofembodiments of the present invention can be that the overall shapes ofthe matrices formed can be complex.

The method may include the step of distributing or seeding a bioactiveagent such as cells in the matrix. This may be performed after thevolume blanking arrangement is removed, for example by inserting thebioactive agent into the voids left by the removed volume blankingarrangement. However, preferably, bioactive agent, particularly cells,are seeded in the matrix by mixing the cells with the hardenable liquidbefore it hardens, for example before the liquid is contacted with thevolume blanking structure. If the matrix is to be a tissue engineeringscaffold, it may be advantageous to include a cell growth factor.Typically, this may be included as part of the volume blankingstructure, which transfers or is transferred to the matrix beforeremoval of the volume blanking structure.

Advantageously, the hardened material is a biocompatible material. Abiocompatible material is considered to be any material which is notexcessively harmful or toxic to living cells or tissue, i.e. non-toxicin the intended environment of use. The material may be inert, or may bedegradable by living cells or tissue, for example by enzymes produced byliving cells or tissue. The material may be suitable for direct implantto a mammalian body. The biocompatible material may be suitable for useas a scaffold for growth of cells either on or within the material asmentioned above. Suitable materials include biologically derivedsubstances such as alginate, collagen, etc., and synthetic materialssuch as heat-softening materials or thermoplastics, etc. Preferably,these will be inert, or will not give rise to excessively toxicdegradation products.

Suitable hardened materials may have a structure which allows thecontrolled release of bioactive agents or substances such aspharmaceuticals, hormones, growth factors, cytokines, antibodies,nucleic acids such as DNA, isolated cell organelles such asmitochondria, killed cells, and the like.

Additionally or alternatively, living cells may be encapsulated in thematrix of hardened material. These cells may be eukaryotic orprokaryotic. In this case the matrix may support exchange of proteins,nutrients, oxygen, secreted molecules and waste products between thecells and a medium surrounding and/or penetrating the hardened matrix.

In this way, the hardened material may act as a tissue engineeringscaffold, supporting growth of the cells. Structures containing livingcells may be cultured in vitro or implanted directly into a patient.When cultured in vitro, the scaffold may be degraded when the cells haveformed an integral mass (e.g. cells and extra cellular matrix) or bodyand when physical support from the scaffold is no longer required.Degradation may be by auto-degradation, or may be caused by adegradative agent such as an enzyme, which may be added exogenously orproduced by the cells within the structure. For example, alginate matrixcan be degraded by exposure to sodium ions or by lyases. An alternativemethod of degradation could involve using antibodies or antibodyfragments. The hardenable material may be chosen appropriately,depending on the intended use, e.g. whether the scaffold is to bedegraded prior to implantation or not.

Suitable combinations of hardenable liquids and hardening agents arewell known in the art. For example, alginate, e.g. sodium alginate canbe cross-linked by calcium ions into a suitable biocompatible material.Accordingly, the hardenable liquid may contain sodium alginate, and maybe hardened by contact with a calcium salt, such as calcium chloride, asthe hardening agent. Another possible combination of components includesacid soluble collagen which cross-links to form a hydrogel when exposedto sodium hydroxide and/or when heated to a temperature of above around4° C. up to around 37° C. Another possible combination is a mixture offibronectin and fibrinogen dissolved in urea which forms a solid whenexposed to a solution of hydrochloric acid/calcium chloride. In generalterms, any natural or synthetic polymers which, for example, arecross-linkable and which are biocompatible (preferably also duringcross-linking or polymerisation) may be used. Combinations of hardenableliquids may be used, e.g. a mixture of alginate and collagen.

The hardenable liquid may include structurally modified molecules. Forexample, alginate may be used where the alginate is modified to includea peptide, e.g. a pentapeptide including for example an RGD sequenceattached to the alginate molecules in order to provide cell attachmentlocations.

The hardening agent is not limited to chemical compounds, although thismay be preferred. The hardening agent may, for example, bring about atemperature change in the hardenable liquid, e.g. it may heat thehardenable liquid to bring about a chemical alteration in the hardenableliquid (e.g. polymerisation or cross-linking).

The hardenable liquid may further contain one or more biologicallyactive agents. These active agents may be active molecules such asenzymes, growth factors, hormones, cytokines, antibodies, nucleic acids,killed cells, isolated cellular organelles, etc. Additionally oralternatively, the biologically active agents may include live cells. Ifthe hardened matrix is required to contain a uniform distribution of abiologically active agent, then the active agent may be homogeneouslymixed into the hardenable liquid, consequently being uniformlydistributed through the structure of the resultant hardened matrix.

The volume blanking structure may be an arrangement or structure formedof one or more (preferably more than one) volume blanking elements. Inthis preferred case, the interconnected spaces may be intersticesbetween adjacent volume blanking elements. Typically, these elements arepacked so that at least some adjacent elements touch. This packing maybe in a suitable vessel such as a tube. In this case, the packedelements may be supported in the tube by a removable sealing member. Itis clearly desirable to form the hardened matrix in a vessel in order tocontain the hardenable liquid. However, the vessel may also provide anexternal limit to the shape of the volume blanking arrangement andtherefore provide the overall shape of the hardened matrix.

The volume blanking structure is typically solid in the sense that itmay be self-supporting. Of course, the volume blanking structure may befurther supported by a vessel, such as referred to above, to furthersupport its shape.

The volume blanking elements are typically solid units, but they mayalternatively be gaseous or liquid (e.g. bubbles or droplets). They maybe hollow. They may be small enough to move relative to each other inthe volume blanking structure if disturbed.

Typically, the volume blanking elements have an average size of 500 μmor less or, more preferably, 100 μm or less. This average size ispreferably more than 1 μm and even more preferably more than 2 μm. Thesize distribution of volume blanking elements may be polymodal, e.g.bimodal. For example, there may be an array of larger volume blankingelements with smaller volume blanking elements. This is discussed inmore detail below.

Clearly, for matrices of a useful size, there will be very many volumeblanking elements used. This will provide very many interconnectedspaces into which the hardenable liquid may flow. Advantageously, afterremoval of the volume blanking elements, the matrix will therefore havea very high internal surface area (i.e. the surface area of the voids).

Preferably, the voids have an average size in the same range as thatdefined for the volume blanking elements, above. Of course, the voidsmay change size after removal of the volume blanking elements.

The preferred pourability of the volume blanking elements means thatthey may be poured into a vessel which can define, in part, an overallshape for the matrix. Thus, the small size of the volume blankingelements means that complex overall shapes, such as the shapes ororgans, can be replicated.

The method may be carried out by first mixing the hardenable liquid withthe volume blanking elements and then subsequently pouring the mixtureinto a vessel or mould.

Typically, each volume blanking element may be a bead. Each bead may bespherical or approximately spherical in shape. However, other suitableshapes may be envisaged, typically rounded shapes such as ellipsoidal orpebble-shape. There are known methods for production of beads of thepreferred size. Such methods can give beads of narrow size distribution.See, for example, New Approaches to Tablet Manufacture. Dr. MarshallWhiteman, Phoqus. European Pharmaceutical Review, Vol. 4, Issue 3,Autumn 1999, and Cowley M, 1999, Powder Coating: Assessment of componentbeing coated: A practical guide to equipment, processes and productivityat a profit, pp. 13-31.

If, as is preferred, the volume blanking structure is to be removablefrom the hardened matrix, then this places a constraint on the materialswhich may be used for the volume blanking structure. Preferably, thematerial is a solid which is soluble in a biocompatible solvent. It ispreferred that the material does not dissolve immediately on contactwith the hardenable liquid, since the volume blanking arrangement shouldgive some mechanical integrity to the hardenable liquid as it hardens. Asuitable material for the volume blanking structure is a soluble sugarsuch as glucose. The material of the volume blanking structure may becapable of sublimation. The material may be biological feedstock such ascarbohydrate, protein, fat or it may be enzymatically degradable. Inthis case, the material would be useful for culturing and growing cellswhich are seeded in the matrix.

The volume blanking structure may include, e.g. collagen, alginate orsimilar hardened materials. The volume blanking structure may includebone-like materials, such as hydroxyapatite (HA). In that case, thehardened matrix may be a tissue engineering scaffold for bone tissue.Part of the volume blanking structure (e.g. the HA) can then stay withinthe matrix to become part of the final engineered tissue.

Typically, the hardening agent is formed as a layer on at least some ofthe volume blanking elements. An advantage here is that the hardeningagent will come into contact with the hardenable liquid before theremainder of the volume blanking element.

The hardening agent layer may have a protective layer formed over it,e.g. an enteric layer. This protective layer is adapted to dissolve at apredetermined rate in the hardenable liquid. This can delay the exposureof the hardening agent to the hardenable liquid. In this way, hardeningof the hardenable liquid can be delayed up until all of the volumeblanking structure has been contacted with hardenable liquid. In apreferred embodiment, the solubility of the protective layer in thehardenable liquid may be dependent on pH. In this way, the dissolutionof the protective layer may be triggered by a change in pH of thehardenable liquid.

The volume blanking elements may further include a cell growth factorlayer. This may be above or below the hardening agent layer, dependingon when in the hardening process it would be suitable for the growthfactor to be released. Typically, the cell growth factor layer will beunderneath the hardening agent layer, thereby to release the growthfactor layer substantially after hardening of the hardenable liquid hasoccurred.

In some preferred embodiments, the formation of the volume blankingstructure includes the formation of one or more selected regions withinthe arrangement with different concentrations of hardening agent to theremainder of the arrangement. An effect of such concentration ofvariations can be to affect the hardening of the hardenable liquid inthose regions. In order to accurately construct such regions in thearrangement, each selected region may be separated from the remainder ofthe structure or arrangement by a retaining surface, such as by asoluble film. This allows the volume blanking structure to be formedwith accurate distribution of concentration of hardening agent.

Each selected region with different concentration of hardening agent maybe an elongate region extending through the structure. Preferably, theconcentration of hardening agent in such regions is insufficient toharden the hardenable liquid placed in the interconnected spaces in suchregions. An effect of this can be that the matrix includes regions ofnon-hardened liquid. In the case where this liquid is subsequentlyremoved, the matrix will include non-filled spaces corresponding tothese selected regions. In this way, the overall internal shape of thematrix may be controlled by controlling the concentration distributionof hardening agent through the arrangement. These regions can be formedso as to define vessels or chambers within the hardened matrix. In thisway, the complex internal shapes of organs such as the liver, kidney,heart, etc. can be mimicked. Of course, in the case of mimickery of suchan organ, the matrix may preferably be seeded with suitable cells (forexample, cells from such an organ from the patient of interest) andother suitable bioactive substances. Of course, the term “organ” is notlimited to these described body parts, but is applicable to other bodyparts such as skin, bone, body lumens such as blood vessels, parts ofthe gastro-intestinal tract, etc.

As mentioned above, the volume blanking structure may include apolymodal size distribution of volume blanking elements. Significantlylarger (e.g. greater than 1 mm in size) volume blanking elements may beincluded. Once dissolved away, these would leave large pores in thematrix. Subsequently, these larger pores may be filled (e.g. byinjection) with a mixture of hardenable liquid and volume blankingelements. Typically, this method allows a main matrix to be formed andseeded with a first cell type (mixed with the hardenable liquid). Thenone or more of the large pores may be filled with matrix seeded with asecond cell type (mixed with the injected hardenable liquid). In thisway, islets of a second cell type may be formed in a matrix of a firstcell type. Of course, this is not limited to two cell types. Three ormore may be used. Furthermore, the larger pores may have predeterminedshapes, e.g. rod-shaped, dependent on the shapes of the larger volumeblanking elements used.

Furthermore, the internal surface of a film used to separate a selectedregion from the rest of the matrix may be used as a guide surface forthe formation of a sheet or preferably a tube of hardened material.Typically this, e.g. tube is seeded with cells of, e.g. smooth muscletype. Typically, the guide surface will be in the form of an internalsurface of a tube.

Preferably, the method further includes the steps of:

-   -   providing a body of hardenable liquid (e.g. the hardenable        liquid described above, or a different one) in contact with a        guide surface for the formation of the layer,    -   relatively moving a regulator member and said guide surface with        a gap between them so that a portion of said body of hardenable        liquid is exposed on said guide surface as a layer of        predetermined thickness thereon,    -   causing hardening of the layer of hardenable fluid thus formed        (e.g. by a hardening agent), to form the hardened layer on the        guide surface.

Our published International Patent Application WO-02/77336, claimingpriority of UK patent applications 0120815.6 (filed 28 Aug. 2001),0107549.8 (filed 26 Mar. 2001) and 0121995.5 (filed 11 Sep. 2001),discloses methods of forming hardened sheets and tubes. The entirecontent of WO-02/77336 is hereby incorporated by reference into thepresent application, and is referred to below.

For example, the layer of hardenable liquid may be caused to harden bycontacting the layer of hardenable liquid with a fluid (hardening agent)causing hardening thereof. The fluid which causes hardening may beselected from:

-   -   a gas containing a hardening agent for the hardenable liquid,    -   a gas effecting hardening of the hardenable liquid by drying,    -   a liquid comprising a reactive hardening agent, e.g. a        cross-linking agent, for the hardenable liquid, and    -   a liquid effecting hardening of the hardenable liquid by solvent        extraction.

Preferably, the fluid causing hardening is progressively immediatelycontacted with the layer of hardenable liquid as the layer is formed bythe relative movement of the regulator member and the guide surface.

The regulator member may act as a barrier separating the fluid causinghardening from said body of the hardenable liquid. Movement of theregulator member may be caused by flow of the fluid causing hardening.The regulator member need not be solid. It may be, for example, gaseous,e.g. a gas bubble sized appropriately.

The regulator member may be driven by a piston action, e.g. by flow ofthe fluid causing hardening.

The regulator member may a float floating on the hardenable liquid. Itmay be a gas bubble.

The hardening liquid may comprise a plurality of discrete bands ofdifferent solutions, to form a hardened layer having substantiallydistinct sub-layers.

The hardened layer may be seeded with cells, for example. These cell maybe of a different type to those cells (if any) seeded in the matrix. Inthis way, the matrix of hardened material may be formed with tubes ofhardened material extending through it. This is particularly desirablefor mimicking the structure of body parts and organs.

The method may further include the step of locating a further shapinginsert in the hardened matrix. This may be, for example, by forming thevolume blanking structure around one or more inserts having a desirableshape. The inserts may be removable mechanically or by dissolution or bya combination of these (e.g. a mechanically removable skeleton coatedwith a soluble solid layer).

In a particularly preferred embodiment, the insert or inserts are forkedor branched. Particularly, Christmas tree shaped inserts are preferred,i.e. a shape with a main trunk which splits progressively along itslength into finer and finer branches (these branches also branching, asappropriate). This may mimic the cardiovascular system. Two (or more)such inserts may be opposed (branched ends facing each other and/or e.g.overlapping and/or intertwining with each other) in a vessel to allow asuitably shaped matrix to be formed.

Matrices provided according to the present invention may be used in awide variety of ways. In addition to the organ replacement use mentionedabove, matrices may be used as structures containing active agents foruse in therapeutic devices such as transdermal delivery patches andother therapeutic devices such as tablets or implants or gene therapydelivery devices.

Biocompatible hardened matrices may also be used as internal grafts fordelivery of any appropriate active substance directly to an internalorgan, or to a disease or wound site. For example, a matrix containingfactors for the promotion of wound healing, such as pro-angiogenicfactors, may be applied to a section of tissue, such as bowel, topromote knitting together of that tissue after surgery (e.g. surgicalanastomosis). Alternatively, pro-angiogenic factors could be deliveredto the heart, or anti-angiogenic factors to a tumour in this way.

Accordingly, in a second aspect, the present invention provides a matrixof hardened material obtained or obtainable via the method of the firstaspect, including any of the preferred features of the first aspect.

In a third aspect, the present invention provides a matrix ofbiocompatible in vitro hardened material having an array ofinterconnected voids therein, the hardened material having a controlleddistribution of a bioactive agent within its volume, and wherein thematrix is preferably not a sheet or tube. The matrix material may behardened by chemical interaction and/or contain cells within thematerial itself.

Typically, the array of interconnected voids is in the form of a packedstructure of contacting rounded shapes, such as spheres. Preferably, theinterconnected voids are partially separated from each other by nodes ofhardened material, each node having a controlled distribution of thebioactive agent through its thickness.

The bioactive agent may be a pharmaceutical or other bioactive molecule,e.g. a pharmaceutical, enzyme, growth factor, hormone, cytokine,antibody, or nucleic acid, to be delivered to a desired site in a livingorganism, e.g. mammal. Additionally or alternatively, the bioactiveagent may include viable cells, killed cells or isolated cellularorganelles.

Preferably, the matrix includes any of the preferred features describedwith respect to the first aspect.

In a fourth aspect, the present invention provides a tissue growthscaffold including a matrix according to the second or third aspect.Further the invention provides a method of tissue growth, e.g.replacement organ growth, comprising cultivating cells contained in thehardened matrix material and/or cells present in the voids within thematrix.

In a fifth aspect, the present invention provides a replacement organformed or formable using a matrix according to the second or thirdaspects of the invention. The replacement organ may be, e.g., areplacement heart, kidney, liver, etc. The replacement organ may be anin vivo replacement organ, i.e. transplanted into a patient, or it maybe an ex vivo organ, such as an organ assist device, to be locatedoutside the body, such as a liver assist device.

In a sixth aspect, the present invention provides a bioreactor includinga matrix according to any one of the second, third or fourth aspectsdisposed in a vessel, the bioreactor further including means for flowingcell culture medium along the vessel and through the matrix. Preferably,the vessel is the vessel in which the matrix was formed.

Typically, the bioreactor also includes means for flowing cell culturemedium through the matrix.

In a preferred embodiment, the hardened matrix is formed in a vessel asdescribed with respect to a preferred feature of the first aspect. Thisvessel preferably is part of the bioreactor, so that the hardened matrixneed not be removed from the vessel before use in the bioreactor. Thiscan maintain the sterility of the hardened matrix and improves thesafety of the bioreactor.

The bioreactor typically comprises a chamber containing a culture ofcells to which a flow of cell culture medium is supplied. The flow ofmedium may for example be continuous or intermittent.

The bioreactor may comprise one or more fluid inlets or outlets forsupply of culture medium to the hardened matrix. Furthermore, it maycomprise one or more ports for probes for measuring conditions such aspH, CO₂ content, oxygen content, etc. in the bioreactor. In a preferredembodiment, this bioreactor can support a flow of culture medium alongthe full length of and/or throughout the hardened matrix.

An important aspect of the bioreactor is that cell culture medium canflow from one end of the matrix to the other. In this sense, it ispreferred that the voids within the matrix are interconnected, sincethis allows flow from one void to the next, promoting easy flow.

Another advantage of maintaining the hardened matrix in the vessel isthat the matrix may be relatively delicate and sensitive to handling.Handling has the potential to damage the matrix itself or the cells tobe cultured.

In a seventh aspect of the invention, there is provided a method offorming a predetermined shape of a hardened material including the stepsof: contacting a hardenable liquid with a mould defining, at least inpart, the predetermined shape, wherein a contacting surface of the mouldincludes a hardening agent; and allowing the hardenable liquid to hardenby chemical interaction with the hardening agent to form thepredetermined shape.

Preferably, the mould includes a vessel and at least one removableinsert. Typically, the removable insert is removed by dissolving it orby partially dissolving it and mechanically removing it. The removableinsert may be formed of similar materials as described with respect tothe volume blanking structure of the first aspect.

This aspect of the invention is similar to the first aspect of theinvention in the sense that the method may be used to give a desirableinternal shape to a hardened material. In particular, this aspect of theinvention may allow the formation of hardened materials with complexinternal shapes, for example one or more internal space in the hardenedmaterial. These shapes may mimic the shape of body parts. For example,they may mimic the shape of vessels, valves (e.g. heart valves) ororgans or parts of organs such as the heart, bones, etc.

The hardenable liquid is preferably the same as that used with respectto the first aspect. This aspect of the invention preferably includesany preferred feature as described with respect to any of the otheraspects of the invention. In particular, a preferred embodiment of theinvention combines the first and seventh aspect to give a method forproducing a matrix of hardened material of predetermined shape.

A further aspect of the present invention provides a hardened matrix ora hardened material of predetermined shape obtained or obtainable by anyof the methods of the previous aspects.

INTRODUCTION OF THE DRAWINGS

Preferred embodiments of the present invention will now be described byway of example only, with reference to the accompanying drawings, inwhich:

FIG. 1 shows a schematic sectional view of a first embodiment of thepresent invention.

FIG. 2 shows a schematic view along the line A-A′ in FIG. 1.

FIG. 3 shows a schematic, modified, enlarged view of a part of thepacking arrangement of FIG. 1.

FIG. 4 shows a schematic sectional view of a bead for use in anembodiment of the present invention.

FIG. 5 shows a schematic sectional view of a hardened matrix accordingto an embodiment of the present invention.

FIG. 6 shows a schematic sectional view of a second embodiment of thepresent invention.

FIG. 7 shows a schematic view along the line B-B′ in FIG. 6.

FIG. 8 shows a schematic sectional view of a bioreactor according to anembodiment of the present invention.

FIG. 9 shows a schematic sectional view of a hardened matrix accordingto another embodiment of the invention.

FIG. 10 is a sectional view of a hardened matrix formed according toanother embodiment of the invention.

FIG. 11 is a sectional view of a hardened matrix formed according toanother embodiment of the invention.

FIG. 12 is a sectional view of a forming apparatus for forming ahardened material in a predetermined shape according to anotherembodiment of the invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

FIG. 1 shows a schematic sectional view of an apparatus for producing ahardened matrix according to an embodiment of the invention. In FIG. 1,a tubular vessel 10 has a sealing plate 12 located at its lower end.Sealing plate 12 is provided to ensure that liquid in the tube 10 abovesealing plate 12 does not leak out of the lower end of tube 10. Sealingplate 12 is removable. It may, for example, be a plunger, capable ofsliding upwards or downwards within the tube 10 (which may be aconverted syringe).

An array of discrete beads 14 is packed within tube 10. The array ofbeads is an example of a volume blanking structure. It is to be notedhere that the schematic packing arrangement as shown in FIG. 1 is veryregular. In practical embodiments of the invention, it is likely thatthe packing of beads 14 will be more or less random. This is becausebeads 14 are typically small, for example around 50 μm in diameter.Beads 14 may be packed in tube 10 simply by pouring the beads into tube10. More sophisticated alternate packing arrangements are describedbelow.

As will be clear to the skilled person, the schematic square packing ofbeads 14 in FIG. 1 is unlikely to occur in practice. However, for now,this schematic arrangement serves an illustrative purpose.

FIG. 2 shows a schematic view along line A-A′ in FIG. 1. This sectionalview from above shows that the beads 14 substantially fill the vessel 10in the width direction. If necessary, plate 12 supports the beads 14.

Beads 14 are typically of rounded shape, as illustrated in the drawings.Preferably, they are spherical. FIG. 3 shows a schematic enlarged viewof some packed beads 14. It is to be noted here that, on a small scale,the beads 14 will tend to be relatively closely packed, as illustratedin FIG. 3. It is the long range order of the beads 14 which will tend tobe relatively random. Between adjacent beads 14 are interstitial spaces16. If the beads are approximately spherical (as in this embodiment),then interstitial spaces 16 are interconnected. Thus, the interstitialspaces in the packed beads arrangement define flow paths through thepacked beads arrangement.

It is preferred that beads 14 are of similar size to each other. Thisgives rise to similarly sized interstitial spaces. However, of course,in practical embodiments, there will be some size distribution of beads14. For this reason, there will be some size distribution ofinterconnected spaces 16.

FIG. 4 shows a schematic sectional view of a typical bead 14. Bead 14includes a core 20 of soluble material such as glucose. This has acoating 22 of a cell growth factor. On coating 22 is a layer 24 ofhardening agent, in this case calcium chloride. The bead 14 has an outerprotective coating 26.

Once the beads 14 have been packed within tubular vessel 10, ahardenable liquid (not shown) is poured into tubular vessel 10 to fillthe spaces 16 between beads 14. The hardenable liquid in this case isalginate. A suitable volume of liquid is used such that there is littleor no excess liquid above or below the packed beads arrangement.

It should be noted here that the beads could be poured into thehardenable liquid. The beads would then pack themselves into aself-supporting structure (helped by the vessel) and thus blank out theliquid from the volume occupied by the bead bodies.

Protective coating 26 on each bead dissolves at a predetermined rate inthe hardenable liquid. This protective coating 26 prevents immediateexposure of the hardenable liquid to the hardening agent layer 24. Thus,the liquid has time to fill all the available spaces between beads 14.The protective layer 26 may have a dissolution rate dependent on the pHof the hardenable liquid. Thus, the pH of the hardenable liquid may bealtered after pouring into the vessel (e.g. by adding suitable acidic oralkaline substances to the liquids) in order to trigger dissolution ofprotective layer 26. Of course, the allowable range of alteration of pHof the hardenable liquid will depend on the effect of pH on biologicalagents contained in the hardenable liquid.

Once the protective layer 26 has dissolved, the alginate comes intocontact with the calcium chloride layer. This has the effect of rapidlyhardening the alginate. Typically, the thickness of the calcium chloridelayer is tailored to the volume of alginate which it is estimated willcome into contact with bead 14.

Beads 14 may be made by known methods of spray forming. Initially, theglucose core 20 is formed and this is subsequently coated by layers 22,24, 26 in a continuous process. Careful control of the spray formingconditions can lead to a uniform size distribution of beads 14 and alsouniform distributions of thicknesses of layers 22, 24, 26. See, forexample, New Approaches to Tablet Manufacture. Dr. Marshall Whiteman,Phoqus. European Pharmaceutical Review, Vol. 4, Issue 3, Autumn 1999,and Cowley M, 1999, Powder Coating: Assessment of component beingcoated: A practical guide to equipment, processes and productivity at aprofit, pp. 13-31.

Once the alginate is hardened by cross-linking due to interaction withthe calcium ions in the calcium chloride layer 24, the cell growthfactor layer 22 is exposed within the hardened alginate matrix. This canbe allowed to leach into the hardened alginate matrix as desired. Thiscan lead to a desirable concentration gradient in growth factorconcentration within the hardened alginate. Since the glucose core 20 isrelatively benign in biological terms, the glucose core 20 can beallowed to remain in place for some time while the growth factor 22leaches into the hardened alginate matrix.

Subsequently, the glucose core 20 may be removed by passing waterthrough the hardened alginate matrix to dissolve the glucose. Once theglucose core has been removed, the hardened alginate matrix containsvoids where the beads 14 were located. A schematic hardened alginatematrix 30 is illustrated in FIG. 5. This is a sectional view. “Hardened”alginate is relatively soft and gel-like. The view shown in FIG. 5 showsupper 32 and lower 34 portions of solidified alginate not containingvoids. The remainder of the alginate matrix consists of a network ofinterconnected hardened alginate nodes 36. Since the spaces 16 in thepacked bead arrangement were interconnected, the hardened alginate isinterconnected since this has replaced the spaces 16. Of course, atypical sectional view will not show all of the interconnections betweenthe various hardened alginate nodes 36. However, some of theseconnections 38 are illustrated in FIG. 5.

Since, in the packed beads arrangement, the beads abut each other, theresultant voids 40 left by the beads 14 are interconnected with eachother (since the alginate liquid occupied only the space not occupied bythe beads 14). This gives a hardened matrix 30 with an advantageousstructure. The interconnected voids 40 provide flow paths for, e.g.culture medium through the hardened matrix 30.

In this preferred embodiment, living cells from a patient such as apatient's liver cells are mixed with the alginate liquid. Alginateliquid is biocompatible with liver cells. It must be ensured, of course,that the protective coating 26 is made of a material which will not harmthe liver cells in the alginate liquid. A uniform distribution of cellswithin the alginate liquid will give rise to a substantially uniformdistribution of cells within the alginate liquid which will give rise toa substantially uniform distribution of cells within the hardenedalginate matrix 30. The provision of growth factor in the hardenedalginate matrix promotes the growth of the cells. Preferably, the cellsare cultured to grow and produce extra cellular material. The alginatematrix may be slowly consumed during this process. In this way, thecells replace the alginate matrix with their own tissue scaffold. Thiscan improve the rigidity and biocompatibility of the arrangement.

FIG. 6 shows a schematic sectional view of a further preferredembodiment of the present invention. FIG. 6 is similar to FIG. 1 in thatis shows a tubular vessel 10 with a packed arrangement of beads 14located above a sealing plate 12. These features will not be describedin detail again.

The packing arrangement of beads 14 is more complex in FIG. 6 than inFIG. 1. In FIG. 6, regions of the packing are separated from theremainder of the packing arrangement by tubes formed from soluble films50, extending downwards through the packing arrangement. These films 50define square rod-shaped regions 52, 54 of packed beads which areisolated from the remainder of the packed beads. FIG. 6 also showshorizontal regions 56, 58 of similarly isolated beads, these beingisolated by films 60, 62.

FIG. 7 shows a schematic view along line B-B′ in FIG. 6. This shows anarray of vertically extending square rod-shaped regions which areisolated from the remainder of the packed bead arrangement.

Before or during packing of the main bead arrangement, isolation film50, for example, is selectively packed with beads having no or littlehardening agent layer 24. This film is nevertheless packed with beads inorder to maintain its shape within the packed arrangement. Very thin,flexible films are used since these may be soluble. It would of coursebe possible to use rigid, empty (unpacked) tubes in the same role, butremoval of these tubes from the hardened matrix may damage the matrix.

As will be clear, when the hardenable alginate liquid in poured into thevessel 10, the liquid occupies the spaces 16 between beads 14. Theliquid is also poured down regions 52, 54. However, in these regions 52,54 the alginate does not harden since there is no sufficient availablehardening agent. Once the remainder of the alginate has hardened, thealginate in regions 52, 54 may be removed. Film 50 may then be dissolvedaway. This leaves a hardened alginate matrix containing vertically (andhorizontally in the case of regions 56, 58) extending channels. In thisway, complex internal shapes which mimic the shapes of organs such asthe liver, heart, kidneys may be formed.

Furthermore, in alternative preferred embodiments, regions 52, 54 couldbe filled with beads containing alternative growth factors to theremainder of the beads. These regions may then be filled with analginate liquid seeded with different cells to the cells seeded in theremainder of the alginate liquid used in the rest of the arrangement. Inthis way, complex, cell-differentiated structures may be engineered.

FIG. 8 shows a bioreactor according to a preferred embodiment of thepresent invention. In FIG. 8, an alginate matrix 30 has been formedwithin tubular vessel 10, as described above. This alginate matrix 30 isseeded with cells which can produce a useful biological agent. Hardenedalginate matrix 30 is not removed from tubular vessel 10. Instead,sealing plate 12 has been removed. The upper and lower ends of tubularvessel 10 are filled by sinter plugs 70, 72. These are rigid yet porousplugs which will prevent movement of hardened alginate matrix 30 out oftube 10. The tube 10 is connected to a cell culture circuit (not showncomplete) including cell culture input tube 74 and cell culture exhausttube 76. These are connected to tubular vessel 10 via sealing member 78(e.g. O-rings). In this way, the cells within the hardened alginatematrix may be grown and cultured and their products harvested withoutinvasive and potentially non-sterile removal of alginate matrix 30 fromtubular vessel 10.

FIGS. 6 and 7 show elongate regions of approximately squarecross-section. It is of course possible to make these elongate regionswith rounded, e.g. circular cross-section. As has already beendescribed, these regions can be formed so as to create tubular spaces inthe hardened matrix. These tubular spaces may themselves be filled withan alternate hardened matrix. Alternatively, the internal surfaces ofthe tubular spaces may be coated with a hardened material. This isillustrated in FIG. 9, which shows a hardened matrix 102 with tubularspaces 104, 106 formed in it by the above-described method. The film 50,in this case, has not yet been dissolved away. The film 50 is formed onthe internal surface of the tubular space. A hardened coating 151A isformed on the exposed surface of the film 50. This formation of coating151A may be independent of the matrix, so that only the film 50, actingas a vessel, takes part in the formation of coating 151A.

Coating 151A is a hardened alginate, in the form of a tube. It may beformed in several different ways, as described below.

Methods and apparatuses suitable for forming a thin-walled tube ofhardened material within the hardened matrix such as the tube 151A, aredescribed and illustrated in WO-02/77336 mentioned above, particularlyin FIGS. 1 to 4 and 8 to 14, to which reference should be made.

Another embodiment of the invention is illustrated in FIG. 10. Thisshows a hardened alginate matrix 102 formed within a vessel 200 in a waysimilar to the first embodiment. However, in this case, the volumeblanking beads used had a bimodal size distribution. Most were small buta few were relatively large (around 5 mm). After dissolution of thebeads, large pores 202 were left within the matrix 102, in addition tothe smaller pores (not shown). The matrix 102 is seeded with cells of afirst type by mixing with the hardenable liquid.

Large pores 202 are subsequently filled with a mixture 204 of beads andhardenable liquid, seeded with a second type of cells. These areinjected into the large pores 202 via a needle 206. In this case, it isimportant that the beads include a protective layer to prevent thealginate from hardening immediately (i.e. before injection).

Mixture 204 hardens into secondary matrix 208. Thus, clumps of cells ofa second type may be formed within a surrounding matrix seeded withcells of the first type.

A further embodiment is illustrated in FIG. 11. This shows a hardenedalginate matrix 300 formed substantially in accordance with the firstembodiment within a tubular vessel 302. The matrix is formed around twotree-shaped inserts 304, 306. These are shaped with a similar externalappearance to, e.g. branching blood vessels.

Inserts 304, 306 have an insoluble skeleton 308, 310, e.g. formed frombiocompatible metal wire. On this skeleton is formed a coating 312, 314of a soluble material. Inserts 304, 306 are removable from the hardenedmatrix (once hardened) by dissolving the coatings 312,314 and pullingthe skeleton 308, 310. In this way, the hardened matrix may be formedwith extremely complex shapes as, e.g. tissue growth scaffolds.

FIG. 12 illustrates another embodiment of the present invention. Apredetermined shape 400 of hardened alginate material is formed in amould consisting of a tubular vessel 402 and a pair of inserts 404, 406.The inserts take up a large proportion of the internal space defined bythe tubular vessel 402. The space remaining is the predetermined shapementioned above. Liquid alginate is fed into this space. Alternatively,the inserts may be pushed in after the liquid alginate in located in thevessel 402. Inserts 404, 406 each have a coating 408, 410 of calciumchloride. This contacts the liquid alginate and allows it to harden.Subsequently, the inserts 404, 406 are removed and the predeterminedshape of hardened alginate is removed from the vessel 402.

The shape illustrated mimics (schematically) the shape of a heart valve.The hardened alginate here is a heart valve tissue engineering scaffold.In this embodiment, since the shape has thin walls, there is no need toinclude beads to harden the alginate through its thickness, or to leavevoids.

Only simple apparatus is required to put the present invention intopractice. Sterile, single-use, disposable apparatus suitable forpractising the methods described can be readily produced at low cost.Manipulations of cells and formation of structures according to thepresent invention can thus be performed under sterile conditions atminimum expense and with minimum risk of contamination. Because of thesimplicity of the apparatus required, the methods described herein caneasily be automated.

The above embodiments have been described by way of example only.Modifications of these embodiments, further embodiments andmodifications thereof will be apparent to the skilled person and as suchare within the scope of the present invention.

1. A method of forming a matrix of hardened material, including thesteps of: contacting a hardenable liquid with a volume blankingstructure, the structure having a dispersion of interconnected spacestherein and including a hardening agent, whereby the hardenable liquidoccupies at least some of said spaces in said structure; and allowingthe hardenable liquid to harden by interaction with the hardening agentto form the matrix.
 2. A method according to claim 1 wherein the volumeblanking structure is formed before the hardenable liquid is contactedwith said structure.
 3. A method according to claim 1 wherein thehardening interaction is a chemical interaction.
 4. A method accordingto claim 1 further including the step of removing the volume blankingstructure to leave corresponding voids in the matrix of hardenedmaterial.
 5. A method according to claim 4 wherein the volume blankingarrangement is removed by dissolving it.
 6. A method according to claim1 wherein the matrix of hardened material is a bulk matrix.
 7. A methodaccording to claim 6 wherein the bulk matrix has a three-dimensionalshape and wherein the smallest dimension is not less than 0.5, 1, 2, 5or 10 mm.
 8. A method according to claim 1 including the step ofdistributing or seeding a bioactive agent in the matrix.
 9. A methodaccording to claim 8 wherein the bioactive agent is dispersed in thehardenable liquid.
 10. A method according to claim 1 wherein thehardened matrix is biocompatible.
 11. A method according to claim 1wherein the volume blanking structure is an arrangement of volumeblanking elements and the interconnected spaces are interstices betweenadjacent volume blanking elements.
 12. A method according to claim 11wherein the volume blanking elements are packed so that adjacentelements touch.
 13. A method according to claim 11 wherein the volumeblanking elements have a size in the range 1-100 μm.
 14. A methodaccording to claim 11 wherein each volume blanking element is a bead.15. A method according to claim 14 wherein each bead is approximatelyspherical in shape.
 16. A method according to claim 11 wherein thehardening agent is formed as a layer on at least some of the volumeblanking elements.
 17. A method according to claim 16 wherein thehardening agent has a protective layer formed over it which dissolvesand delays the exposure of the hardening agent to the hardenable liquid.18. A method according to claim 17 wherein the solubility of theprotective layer is dependent on pH and the dissolution of theprotective layer is triggered by a change in pH of the hardenableliquid.
 19. A method according to claim 16 wherein the hardening agentlayer has a cell growth factor layer under it.
 20. A method according toclaim 1 wherein the formation of the volume blanking structure includesthe formation of one or more selected regions within the structure withdifferent concentrations of hardening agent to the remainder of thestructure.
 21. A method according to claim 20 wherein each selectedregion is an elongate region extending through the structure.
 22. Amethod according to claim 20 wherein each selected region is separatedfrom the remainder of the structure by a retaining surface.
 23. A methodaccording to claim 22 wherein the retaining surface is a surface of asoluble film.
 24. A method according to claim 20 wherein theconcentration of hardening agent in each selected region is insufficientto harden the hardenable liquid placed in the spaces in that region. 25.A method according to claim 1 wherein the hardenable liquid is alginate.26. A method according to claim 1 wherein the hardening agent includescalcium ions.
 27. A method according to claim 1 wherein the volumeblanking arrangement includes glucose.
 28. A matrix of hardened materialobtained via the method of claim
 1. 29. A matrix of hardened materialobtainable via the method of claim
 1. 30. A body having a matrix ofbiocompatible material hardened in vitro by chemical interaction and anarray of interconnected voids, the matrix of hardened material having acontrolled distribution of a bioactive agent within it, and wherein thebody is not a sheet or tube.
 31. A body according to claim 30 whereinthe bioactive agent is a pharmaceutical or other bioactive molecule,e.g. a pharmaceutical, enzyme, growth factor, hormone, cytokine,antibody, or nucleic acid, to be delivered to a desired site in a livingmammal.
 32. A body having a matrix of biocompatible in vitro hardenedmaterial and an array of interconnected voids, the matrix of hardenedmaterial having a distribution of bioactive agent in the form of cellswithin it, and wherein the body is not a sheet or tube.
 33. A bodyaccording to claim 30 wherein the array of interconnected voids is inthe form of a packed structure of contacting rounded shapes, such asspheres.
 34. A body according to claim 30 wherein the interconnectedvoids are partially separated from each other by nodes of hardenedmaterial, each node having a controlled distribution of the bioactiveagent through its thickness.
 35. A body according to claim 30 whereinthe bioactive agent is viable cells, killed cells or isolated cellularorganelles.
 36. A tissue growth scaffold including a matrix according toclaim
 28. 37. A method of tissue growth, e.g. organ production,comprising culturing of cells contained within the hardened material ofthe matrix according to claim 28 and/or culturing of cells contained inthe pores of any such matrix.
 38. Tissue, e.g. an organ, grown by themethod of claim
 37. 39. A bioreactor including the matrix according toclaim 28 disposed in a vessel, the bioreactor further including meansfor flowing cell culture medium along the vessel and through the matrix.40. A bioreactor according to claim 39 wherein the vessel is the vesselin which the matrix was formed.
 41. A method of forming a predeterminedshape of a hardened material including the steps of: contacting ahardenable liquid with a mould defining, at least in part, thepredetermined shape, wherein a contacting surface of the mould includesa hardening agent; and allowing the hardenable liquid to harden bychemical interaction with the hardening agent to form the predeterminedshape.
 42. A method according to claim 41 wherein the mould includes avessel and at least one removable insert.
 43. A method according toclaim 42 wherein the removable insert is removed by dissolving it or bypartially dissolving it and mechanically removing it.